ABSTRACT Muscular dystrophies associated with mutations in the dystrophin glycoprotein complex (DGC) are characterized by striated muscle degeneration, muscle weakness and fatigue, cardiomyopathy and early death. Due to lack of understanding of the precise causal mechanisms, there are no cures available for muscular dystrophy and current treatments only modestly alter disease progression and function. The long term goal is to understand the molecular mechanisms of cardiac and skeletal muscle dysfunction in muscular dystrophy and target those mechanisms for improving muscle function. Considerable past research effort has focused on understanding the structural role of the DGC in stabilizing the sarcolemma. The potential critical role of the DGC in striated muscle cell signaling is less understood. Nitric oxide (NO) signaling is required in muscle for regulating muscle blood flow during muscle activity and NO synthesis is disrupted in dystrophic muscle. However, very little is known regarding how the activity of NOS is acutely activated by muscle contraction, and what mechanistic role the DGC has in regulating NOS activity. While much of the field has focused on the role of dystrophin in scaffolding nNOS to the sarcolemma in skeletal muscle, this scaffolding hypothesis does not explain how NOS activity is regulated by muscle contraction. Furthermore, the DGC does not physically interact with nNOS in cardiac muscle and the role of the DGC in regulating cardiac NOS is virtually unstudied. The objective this application is to understand the mechanisms of how NOS activity is regulated by mechanical activity of the muscle and demonstrate how the DGC participates as a mechanosensor in this signaling pathway, and then provide preclinical support for pharmacologically restoring the activation of NO signaling to restore dystrophic muscle function. Towards this end, we have developed an innovative model system using mouse model and patient derived cardiac muscle cells to study the mechanisms of acute mechanical activation of NO signaling in striated muscle. Our preliminary data show mechanoregulation of the AMPK signaling pathway is required for regulating nNOS activity and challenges the current model that regulation of nNOS activity requires a physical interaction of dystrophin and NOS. The proposed work will 1) Dissect the mechanistic role of the DGC in mechanoregulation of AMPK-NO signaling using cardiac muscle cells from mouse models and human patients as models of striated muscle 2) Determine the role of DGC dependent mechanoregulation of AMPK-NO signaling in regulating both cardiac AND skeletal muscle blood flow and function. 3) Test whether acute activation of AMPK has acute therapeutic benefit in dystrophic cardiac and skeletal muscle and exercise induced fatigue by activating NO signaling. By improving our understanding of how nitric oxide is regulated by muscle activity and the DGC in muscle, targeted therapies can be developed to restore NO signaling and reduce muscle fatigue and weakness, and cardiomyopathy in muscular dystrophies.